Method of producing L-amino acids

ABSTRACT

Provided is a method of producing L-amino acids by using a recombinant coryneform microorganism in which the expression of a target gene is weakened by using a gene transcription inhibition method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/498,124, filed Sep. 26, 2014, which claims the benefit of KoreanPatent Application No. 10-2013-0121090, filed on Oct. 11, 2013, andKorean Patent Application No. 10-2014-0091307, filed on Jul. 18, 2014,in the Korean Intellectual Property Office, the disclosures of each ofwhich are incorporated herein in their entirety by reference.

BACKGROUND

1. Field

One or more embodiments of the present invention relate to a method ofproducing L-amino acids by using a gene transcription inhibition method.

2. Description of the Related Art

Pyruvate, which is produced through glycolysis of various carbon sourcesin coryneform microorganisms, is converted into aspartate viaoxaloacetate. The aspartate is converted into amino acids such asthreonine, methionine, isoleucine, and lysine through variousbiosynthetic pathways (FIG. 1). Therefore, the expression of genes,located at each branch point in the amino acid biosynthetic processes,may be inhibited to decrease byproduct production and increase targetamino acid production.

As described above, to develop a microorganism strain, which is capableof high-potency production of target materials by using geneticengineering and metabolic engineering, the expression of genes relatedwith various metabolic processes of a microorganism needs to beselectively controlled. Recently, a technology for weakening geneexpression, which is called “artificial convergent transcription,” wasreported (Krylov et al., J Mol Microbiol Biotechnol, 18:1-13, 2010). Theartificial convergent transcription is a technology for weakening theexpression of a target gene by inserting a promoter into a downstreamregion of a transcription terminator of the target gene so that theopposite direction of the promoter causes a collision of RNA polymerasecomplexes derived from each promoter during transcription.

The inventors developed a technology to selectively inhibit theexpression of a target gene in the presence of acetate by inserting anacetate-inducible promoter in a direction opposite to the target genetranscription, and effectively applied the technology to inhibit theexpression of genes located at branch points in a coryneformmicroorganism. Then the inventors verified to provide the coryneformmicroorganism of producing L-amino acid with high yield by using thetechnology and completed the present invention.

SUMMARY

The purpose of the present invention is to provide a method of producingL-amino acids by using an acetate-inducible promoter to inhibittranscription of a target gene.

One embodiment of the present invention provides a method of producingL-amino acids, the method including

1) culturing a recombinant coryneform microorganism capable of producingL-amino acids, wherein the recombinant coryneform microorganism istransformed by inserting an acetate-inducible promoter to the downstreamof a stop codon of a target gene in a chromosome; and

2) adding acetate during the culturing to weaken expression of thetarget gene, and to strengthen the L-amino acids production capabilityof the recombinant coryneform microorganism.

BRIEF DESCRIPTION OF THE DRAWINGS

These and/or other aspects will become apparent and more readilyappreciated from the following description of the embodiments, taken inconjunction with the accompanying drawings in which:

FIG. 1 shows branch points of the amino acids biosynthesis process in acoryneform microorganism;

FIG. 2A is a schematic diagram showing inhibition of aceE geneexpression by inserting an aceA gene promoter between the stop codon andthe transcription terminator upstream of the aceE gene in a directionopposite to the direction of the aceE gene transcription; and

FIG. 2B is a schematic diagram showing inhibition of aceE geneexpression by inserting an aceA gene promoter to the downstream of thetranscription terminator in a direction opposite to the direction of theaceE gene transcription.

DETAILED DESCRIPTION

Reference will now be made in detail to embodiments, examples of whichare illustrated in the accompanying drawings, wherein like referencenumerals refer to like elements throughout. In this regard, the presentembodiments may have different forms and should not be construed asbeing limited to the descriptions set forth herein. Accordingly, theembodiments are merely described below, by referring to the figures, toexplain aspects of the present description. Expressions such as “atleast one of,” when preceding a list of elements, modify the entire listof elements and do not modify the individual elements of the list.

Hereinafter, the present invention is described in detail.

One embodiment of the present invention provides a method of producingL-amino acids, the method including

1) culturing a recombinant coryneform microorganism capable of producingL-amino acids, wherein the recombinant coryneform microorganism istransformed by inserting an acetate-inducible promoter to the downstreamof a stop codon of a target gene in a chromosome; and

2) adding acetate during the culturing to weaken the expression of thetarget gene, and to strengthen the L-amino acids production capabilityof the recombinant coryneform microorganism.

The term “acetate-inducible promoter” used herein refers to a promoterhaving gene expression-inducing activity in the presence of acetate.

In a coryneform microorganism, acetate is converted by acetate kinase(ackA, NCgl2656) and phosphotransacetylase (pta, NCgl2657), or bysuccinyl-CoA:acetate CoA-transferase (actA, NCgl2480), into acetyl CoA,and then metabolized by isocitrate lyase (aceA, NCgl2248) in aglyoxalate cycle. In Escherichia coli, acetate is converted into acetylCoA by acetyl-CoA synthetase (acs, b4069) (Gerstmeir et al., JBiotechnol, 104:99-122, 2003). The expression of the mentioned genesinvolved in the acetate metabolism is induced in the presence ofacetate. Therefore, when the promoters of the genes are used, theexpression of the gene may be specifically induced in the presence ofacetate.

Acetate-inducible promoters include a promoter of a gene encodingisocitrate lyase (aceA, NCgl2248) or a promoter of an operon of a geneencoding acetate kinase (ackA, NCgl2656) and a gene encodingphosphotransacetylase (pta, NCgl2657), which is an upstream promoter ofthe pta gene. More specifically, among the acetate-inducible promotersdescribed above, the promoter of the aceA gene is represented bynucleotide sequence of SEQ ID NO: 1 and includes 486 base pairs in theupstream of the aceA gene and 36 base pairs from an N-terminal of anopen reading frame (ORF).

The upstream promoter of the pta gene, which is anotheracetate-inducible promoter, is represented by nucleotide sequence of SEQID NO: 2 and includes 340 base pairs in the upstream of the pta gene.

In addition, it is obvious that any promoter capable of inducing atarget gene expression by acetate may be included in the scope of thepresent invention. For example, the acetate-inducible promoters mayinclude a nucleotide sequence including the nucleotide sequence of SEQID NO: 1 or 2, or including a conserved sequence of the nucleotidesequence of SEQ ID NO: 1 or 2, and one or a plurality of nucleotides(specifically 2 to 20, more specifically, 2 to 10, further morespecifically, 2 to 5 nucleotides, depending on the steric conformationof amino acid residues of a protein) that are substituted, deleted,inserted, added, or inversed at one or more locations. As long as thefunction of the inducible promoter is maintained or strengthened, it mayinclude a nucleotide sequence has more than 80% homology with thenucleotide sequence of SEQ ID NO: 1 or 2, specifically by more than 90%,more sepecifically by more than 95%, further more specifically by morethan 97%. As long as the function of the inducible promoter ismaintained, the substituted, deleted, inserted, added, or inversednucleotide sequence may include a spontaneous mutant sequence or even anartificial mutant sequence.

The term “homology” used herein refers to the identity between twodifferent nucleotide sequences. Homology may be determined by a methodknown in this art by using a BLAST 2.0 software program which calculatessuch parameters as score, identity, and similarity. However, the methodof determining homology is not limited thereto.

Unless mentioned otherwise herein, the term “upstream” refers to a 5′direction, and the term “downstream” refers to a 3′ direction. Usually,the direction of the transcription proceeding is 5′ to 3′, so that thepromoter position is usually the upstream (5′) of the target gene.

Herein, a target gene in a chromosome may be a gene involved in aprocess of biologically synthesizing amino acids such as threonine,methionine, isoleucine, and lysine from various carbon sources,especially a gene located at a branch point of a biosynthetic pathway.

For example, with respect to lysine, a pyruvate dehydrogenase subunit E1(aceE, NCgl2167) gene which is involved in the conversion of pyruvateinto acetyl CoA, a homoserine dehydrogenase (hom, NCgl1136) gene whichproduces homoserine from aspartate, and aUDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (murE,NCgl2083) gene which uses meso-2,6-diaminopimelate that is a precursorof lysine in somatic synthesis, are located at branch points of thebiosynthetic pathway.

In addition, a dihydrodipicolinate synthase (dapA, NCgl1896) geneinvolved in producing lysine from aspartate is at a branch point withrespect to threonine, and a homoserine kinase (thrB, NCgl1137) geneinvolved in producing threonine from homoserine is at a branch pointwith respect to methionine. With respect to alanine and valine that arepyruvate-derived amino acids, a pyruvate dehydrogenase subunit E1 (aceE,NCgl2167) gene involved in the conversion of pyruvate into acetyl CoA isat a branch point.

Therefore, a target gene may be selected from the group consisting of agene encoding a pyruvate dehydrogenase subunit E1 (aceE, NCgl2167), agene encoding homoserine dehydrogenase (hom, NCgl1136), a gene encodinga UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase(murE, NCgl2083), and a gene encoding dihydrodipicolinate synthase(dapA, NCgl1896), but is not limited thereto.

Specifically, the pyruvate dehydrogenase subunit E1 (aceE, NCgl2167) isone of the protein subunits of pyruvate dehydrogenase complex (PDHC),which is involved in the inflow of pyruvate that is a final metaboliteof glycolysis to a tricarboxylic acid cycle (TCA cycle). Therefore,weakening the expression of the aceE gene may decrease the inflow ofcarbon sources to a TCA cycle and increase the inflow of carbon sourcesto a lysine biosynthetic pathway to increase lysine production.

Homoserine dehydrogenase (hom, NCgl1136) is an enzyme synthesizinghomoserine from aspartate semialdehyde. Since aspartate semialdehyde isone of the intermediate precursors of a lysine biosynthetic pathway,weakening the hom gene activity may decrease the inflow of carbonsources to a homoserine biosynthetic pathway and increase the inflow ofcarbon sources to a lysine biosynthetic pathway to increase lysineproduction.

UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (murE,NCgl2083) uses meso-2,6-diaminopimelate for somatic synthesis. Sincemeso-2,6-diaminopimelate is also used as a precursor for lysinebiosynthesis, weakening of the murE gene activity may decrease theinflow of carbon sources to somatic synthesis and increase the inflow ofcarbon sources to a lysine biosynthetic pathway to increase lysineproduction.

Dihydrodipicolinate synthase (dapA, NCgl1896) is an enzyme which isinvolved in lysine production by using aspartate semialdehyde. Sinceaspartate semialdehyde is one of the intermediate precursors of a lysinebiosynthetic pathway, weakening of the dapA gene activity may decreasethe inflow of carbon sources to lysine biosyntheric pathway and increasethe inflow of carbon sources to a threonine biosynthetic pathway toincrease threonine production.

The term “stop codon” used herein refers to codons that do not encode anamino acid on mRNA, but operate as a signal for termination of proteinsynthesis. Three codons including UAA, UAG, and UGA are usedconventionally as stop codons.

The term “transcription terminator” used herein refers to an GCbase-rich inverted repeat sequence. A transcription terminator forms ahairpin loop to terminate gene transcription.

In the present invention, to weaken the expression of a target gene, asdescribed above, an acetate-inducible promoter may be introduced to adownstream of a stop codon of a target gene, specifically, between astop codon and an upstream of a transcription terminator. Anacetate-inducible promoter may be used to cause an inverse transcriptionof a target gene so that RNA polymerase complexes may conflict with eachother to weaken the expression of a target gene.

The expression of a target gene may be weakened any time of culturing.More specifically the expression of a target gene may be weakened beforeor during culturing.

The term “transformation” used herein refers to introducing a vectorincluding a polynucleotide which encodes a target gene into a host cellso that a protein encoded by the polynucleotide may be expressed in thehost cell. As long as an introduced polynucleotide may be expressed in ahost cell, the polynucleotide may be inserted into the chromosome of thehost cell or exist out of the chromosome. In addition, thepolynucleotide includes DNA or RNA encoding a target protein. As long asthe polynucleotide can be introduced into and expressed in a host cell,the polynucleotide may be introduced in any form.

In an embodiment of the invention, the coryneform microorganism mayinclude microorganisms of genus Corynebacterium, genus Brevibacterium,genus Arthrobacter sp., and genus Microbacterium sp. Examples of thecoryneform microorganism include Corynebacterium glutamicum,Corynebacterium thermoaminogenes, Brevibacterium flavum, Brevibacteriumlactofermentum, and L-amino acid-producing variants prepared therefrom.Specifically, the coryneform microorganism may be Corynebacteriumglutamicum, but is not limited to these examples.

More specifically, the coryneform microorganisms in the presentinvention may include Corynebacterium glutamicum KCCM11016P (FormerAccession NO: KFCC10881, Refer to Korean Patent NO: 10-0159812),Corynebacterium glutamicum KCCM10770P (Refer to Korean Patent NO:10-0924065), and Corynebacterium glutamicum KCCM11347P (Former AccessionNO: KFCC10750, Refer to Korean Patent NO: 10-0073610).

Corynebacterium glutamicum CJ3P may be also included the the coryneformmicroorganisms in the present invention. CJ3P has been developed to havelysine-producing capability by introducing mutation to three genesinvolved in lysine-producing efficiency (pyc(P458S), hom(V59A), andlysC(T311I)) to a parent strain, a wild type Corynebacterium glutamicum(ATCC13032) according to the report by Binder et al. (Binder et al.,Genome Biology, 13:R40, 2012).

In addition, another coryneform microorganism in the present inventionmay be Corynebacterium glutamicum KCCM11222P, which is anL-threonine-producing strain (Refer to Korean Patent NO: 10-1335853).

According one embodiment of the present invention, in all the coryneformmicroorganisms to which a promoter being represented by nucleotidesequence of SEQ ID NO: 1 or SEQ ID NO: 2 was introduced, the L-lysine orL-threonine productivity was increased compared to that of the parentstrain.

With respect to the method provided in the present invention, culturingof a coryneform microorganism may be performed by applying any culturingconditions and culturing method known in this art.

A culture medium, which may be used in culturing of a coryneform strain,may be, for example, the culture mediums described in Manual of Methodsfor General Bacteriology by the American Society for Bacteriology(Washington D.C., USA, 1981).

The carbon sources, which may be used in the culture medium, may includea carbohydrate such as glucose, saccharose, lactose, fructose, maltose,starch, and cellulose, an oil or lipid such as soybean oil, sunfloweroil, castor oil, and coconut oil, a fatty acid such as palmitic acid,stearic acid, and linoleic acid, an alcohol such as glycerol andethanol, and an organic acid such as acetic acid. These substances maybe individually or as a mixture.

The nitrogen sources, which may be used in the culture medium, mayinclude peptone, yeast extract, beef extract, malt extract, corn steepliquid, soybean, and urea, and an inorganic nitrogen source such asammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate, and ammonium nitrate. These nitrogen sources also may beindividually or as a mixture.

The phosphorous sources, which may be used in the culture medium, mayinclude potassium dihydrogen phosphate, dipotassium hydrogen phosphate,and a sodium-containing salt thereof. In addition, a culture medium mayhave to include a metal salt such as magnesium sulfate and iron sulfateneeded for growth. Beside the substances described above, necessarysubstances for growth such as amino acids and vitamins may be used. Inaddition, appropriate precursors may be used in a culture medium. Theraw materials may be added to the culture solution in a batch mode or acontinuous mode during the culturing.

During the culturing of the microorganism, the pH of the culture mediummay be adjusted by adding a basic compound such as ammonium hydroxide,potassium hydroxide, and ammonia, or an acidic compound such asphosphoric acid and sulfuric acid to the culture medium in anappropriate mode. In addition, bubble formation may be repressed byusing an anti-forming agent such as fatty acid polyglycol ester. Tomaintain aerobic conditions, oxygen or an oxygen-containing gas (forexample, air) may be injected to the culture medium. The temperature ofthe culture medium may be usually from about 20° C. to about 45° C.,specifically, from about 25° C. to about 40° C. The culturing maycontinue until a desired amount of an L-amino acid is produced, but anappropriate culturing time may be from about 10 to 160 hours.

With respect to the method provided in the present invention, culturingmay be performed in a continuous mode or a batch mode such as a batchprocess, a fed-batch process, and a repeated fed-batch process. Theseculturing methods are known in the art, and any of the culturing methodsmay be used.

The term “culturing” used herein may include both preparing a culturemedium and the time during growing the microorganisms.

With respect to the method provided in the present invention, the methodmay include further purifying or recovering step. A target L-amino acidmay be purified or recovered from a culture solution by using anappropriate method known in this art according to the method such asbatch culturing, continuous culturing, and fed-batch culturing.

An L-amino acid produced in the culturing of the present invention maybe one selected from the group consisting of threonine, methionine,isoleucine, lysine, valine, and alanine, specifically, lysine orthreonine.

Hereinafter, the present invention will be described in further detailwith reference to examples. These examples are illustrative purposesonly and the scope of the present invention is not limited thereto.

EXAMPLES Example 1: Selection of Acetate-Inducible Promoter

Isocitrate lyase (aceA, NCgl2248) is a key enzyme of a glyoxylate cycle,and a gene encoding isocitrate lyase is expressed in the presence ofacetate. In addition, acetate kinase (ackA, NCgl2656) andphosphotransacetylase (pta, NCgl2657), which are enzymes that areinvolved in an acetate metabolic process, form an operon, and theexpression thereof is strengthened in the presence of acetate. Thepromoter regions of the aceA gene and the pta-ackA operon are alreadyknown (Gerstmeir et al., J Biotechnol, 104, 99-122, 2003).

In Example 1, a promoter of the aceA gene and a promoter of the pta-ackoperon that is an upstream promoter region of the pta gene were selectedto inhibit the transcription of a target gene in the presence ofacetate. Based on the aceA gene registered in the US NIH GenBank (NCBIRegistration NO: NCgl2248), a nucleotide sequence (SEQ ID NO: 1),including 486 base pairs in the upstream of the aceA gene and 36 basepairs from an N-terminal of an open reading frame (ORF), was obtained.In addition, Based on the pta gene registered in the US NIH GenBank(NCBI Registration NO: NCgl2657), a nucleotide sequence (SEQ ID NO: 2),including 340 base pairs in the upstream of the pta gene, was obtained.

Example 2: Preparation of Vector for Inhibiting aceE Gene Expression

A pyruvate dehydrogenase subunit E1 (aceE, NCgl2167) is one of theprotein subunits of pyruvate dehydrogenase complex (PDHC), which isinvolved in inflowing pyruvate that is a final metabolite of glycolysisto a TCA cycle. Therefore, weakening the expression of the aceE gene maydecrease the inflow of carbon sources to a TCA cycle and increase theinflow of carbon sources to a lysine biosynthetic pathway to increaselysine production (Blombach et al., Appl Microbiol Biotechnol,76(3):615-23, 2007).

The aceA gene promoter was inserted to the downstream of the aceE geneso that the transcription from this promoter may occur in a directionopposite to the original direction of the aceE gene transcription toinhibit the expression of the aceE gene selectively in the presence ofacetate (FIG. 2).

Firstly, CLC main workbench software (CLC Bio, Denmark) was used topredict a transcription terminator of the aceE gene. A transcriptionterminator is a GC base-rich inverted repeat sequence and forms ahairpin loop to terminate gene transcription. The result of predicting atranscription terminator of the aceE gene showed that 36 base pairs,from the 21st base pair to the 56th base pair in the downstream from thestop codon of the aceE gene, form a hairpin loop as a transcriptionterminator. Based on this result, two vectors were prepared to insertaceE promoter into either the upstream or the downstream, respectively,of the transcription terminator of aceE gene so that the transcriptionfrom the this promoter may occur in a opposite direction of the originalone.

<2-1> Preparation of pDZ-aceE1-PaceA Vector for Inhibiting aceE GeneExpression

A vector was prepared by inserting an aceA gene promoter to thedownstream of the aceE gene stop codon, which is between the stop codonand the upstream of the transcription terminator.

To obtain a fragment of a Corynebacterium glutamicum-derived aceE gene,the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used asa template to synthesize primers (SEQ ID NOs: 3 and 4), which weredesigned to have an XbaI restriction enzyme recognition site at a 5′ endof the fragment and an SpeI restriction enzyme recognition site at a 3′end of the fragment. A PCR was performed by using the synthesizedprimers to obtain a DNA fragment including 296 base pairs between the2474th nucleotide from the start codon of aceE gene and the 2769thnucleotide, a stop codon of aceE gene. In addition, primers (SEQ ID NOs:5 and 6), which were designed to have an SpeI recognition site at a 5′end of the fragment and an XbaI recognition site at a 3′ end of thefragment were synthesized, and a PCR was performed by using the primersto obtain a DNA fragment including 300 base pairs in the downstream ofthe aceE gene stop codon. PfuUltra™ High-Fidelity DNA Polymerase(Stratagene) was used as a polymerase, and a PCR was performed with 30cycles of denaturing at 95° C. for 30 seconds; annealing at 55° C. for30 seconds; and polymerization at 72° C. for 30 seconds and thenpolymerization at 72° C. for 7 minutes.

The two PCR amplification products and a pDZ vector (Refer to KoreanPatent NO: 10-0924065) for chromosomal introduction that had alreadybeen prepared by cleaving with an XbaI restriction enzyme were cloned byusing an In-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-aceE1vector.

SEQ ID NO: 3: aceE-P1F 5′-ccggggatcctctagacctccggcccatacgttgc-3′SEQ ID NO: 4: aceE-P1R 5′-ttgagactagttattcctcaggagcgtttg-3′SEQ ID NO: 5: aceE-P2F 5′-gaataactagtctcaagggacagataaatc-3′SEQ ID NO: 6: aceE-P2R 5′-gcaggtcgactctagagaccgaaaagatcgtggcag-3′

To obtain a promoter fragment of a Corynebacterium glutamicum-derivedaceA gene, primers which were designed to have an SpeI restrictionenzyme recognition site at a 5′ end and at a 3′ end of the fragment (SEQID NOs: 7 and 8) were synthesized. The PCR was performed using thechromosomal DNA of Corynebacterium glutamicum KCCM11016P as a templateand the synthesized primers to amplify a promoter region of about 500base pairs being represented by a nucleotide sequence of SEQ ID NO: 1.The PCR amplification product and a DNA fragment that was obtained bytreating a pDZ-aceE1 with SpeI restriction enzyme were cloned by usingan In-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-aceE1-PaceAvector.

SEQ ID NO: 7: PaceA-P3F 5′-gtcccttgagactagtagcactctgactacctctg-3′SEQ ID NO: 8: PaceA-P3R 5′-ctgaggaata actagtttcctgtgcggtacgtggc-3′

<2-2> Preparation of pDZ-aceE2-PaceA Vector for Inhibiting aceE GeneExpression

A vector was prepared by inserting an aceA gene promoter to thedownstream of the aceE gene transcription terminator.

To obtain a fragment of a Corynebacterium glutamicum-derived aceE gene,the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used asa template, and primers, which were designed to have an XbaI restrictionenzyme recognition site at a 5′ end of the fragment and an SpeIrestriction enzyme recognition site at a 3′ end of the fragment (SEQ IDNOs: 9 and 10), were used. A PCR was performed by using the synthesizedprimers to obtain a DNA fragment including 294 base pairs between the2538th nucleotide from the start codon of aceE gene and the 62ndnucleotide in the downstream of the stop codon. In addition, primers,which were designed to have an SpeI restriction enzyme recognition siteat a 5′ end of the fragment and an XbaI restriction enzyme recognitionsite at a 3′ end of the fragment (SEQ ID NOs: 11 and 12), were used toperform a PCR to obtain a DNA fragment including 294 base pairs betweenthe 69th nucleotide in the downstream of the aceE gene stop codon andthe 362nd nucleotide. PfuUltra™ High-Fidelity DNA Polymerase(Stratagene) was used as a polymerase, and a PCR was performed with 30cycles of denaturing at 95° C. for 30 seconds; annealing at 55° C. for30 seconds; and polymerization at 72° C. for 30 seconds and thenpolymerization at 72° C. for 7 minutes.

The two PCR amplification products and a pDZ vector for chromosomalintroduction that had already been prepared by cleaving with an XbaIrestriction enzyme were cloned by using an In-fusion Cloning Kit(TAKARA, JP) to prepare a pDZ-aceE2 vector.

SEQ ID NO: 9: aceE-P4F 5′-ccggggatcctctagaggtcccaggcgactacacc-3′SEQ ID NO: 10: aceE-P4R 5′-gagctactagtacgacgaatcccgccgccagacta-3′SEQ ID NO: 11: aceE-P5F 5′-gtcgtactagtagctctttttagccgaggaacgcc-3′SEQ ID NO: 12: aceE-P5R 5′-gcaggtcgactctagacatgctgttggatgagcac-3′

To obtain a promoter fragment of a Corynebacterium glutamicum-derivedaceA gene, primers, which were designed to have an SpeI restrictionenzyme recognition site at a 5′ end and at a 3′ end of the fragment (SEQID NOs: 13 and 14), were synthesized. The PCR was performed using thechromosomal DNA of Corynebacterium glutamicum KCCM11016P as a templateand the synthesized primers to amplify a promoter region of about 500base pairs being represented by a nucleotide sequence of SEQ ID NO: 1.The PCR amplification product and a DNA fragment that was obtained bytreating a pDZ-aceE2 vector with SpeI restriction enzyme were cloned byusing an In-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-aceE2-PaceAvector.

SEQ ID NO: 13: PaceA-P6F 5′-aaaaagagctactagtagcactctgactacctctg-3′SEQ ID NO: 14: PaceA-P6R 5′-gattcgtcgtactagtttcctgtgcggtacgtggc-3′

Example 3: Preparation of Strains in Which aceA Gene Promoter isInserted to Downstream of aceE Gene

The pDZ-aceE1-PaceA and pDZ-aceE2-PaceA vectors prepared in Example 2were introduced respectively by electric pulsing into Corynebacteriumglutamicum KCCM11016P that is an L-lysine-producing strain(transformation method described in Van der Rest et al., Appl MicrobiolBiotechnol, 52:541-545, 1999). The respective strains in which an aceAgene promoter was inserted into the downstream of the aceE gene stopcodon on the chromosome so that the transcription from this promoter mayoccur in a direction opposite to the original direction were selected byperforming a PCR to obtain an L-lysine-producing strain. The selectedstrains were named as Corynebacterium glutamicumKCCM11016P::aceE1-PaceA, and as Corynebacterium glutamicumKCCM11016P::aceE2-PaceA, respectively. Corynebacterium glutamicumKCCM11016P::aceE1-PaceA was internationally deposited in the name ofCorynebacterium glutamicum CA01-2271 to Korean Culture Center ofMicroorganism (KCCM) on Jun. 12, 2013 with Accession Number KCCM11432P.The prepared strains were verified that the nucleotide sequence oftarget region obtained was analyzed by performing a PCR using SEQ ID NO:3 and SEQ ID NO: 6 as primers for KCCM11016P::aceE1-PaceA, and SEQ IDNO: 9 and SEQ ID NO: 12 for as primers for KCCM11016P::aceE2-PaceA.

Example 4: Comparison of Lysine Productivity of Strains in Which aceAGene Promoter is Inserted to Downstream of aceE Gene

Corynebacterium glutamicum KCCM11016P strain which was used as a parentstrain, and Corynebacterium glutamicum KCCM11016P::aceE1-PaceA andCorynebacterium glutamicum KCCM11016P::aceE2-PaceA which were theL-lysine-producing strains prepared in Example 3 were cultured by themethod described below.

Corynebacterium glutamicum KCCM11016P KCCM11016P::aceE1-PaceA, andKCCM11016P::aceE2-PaceA were inoculated respectively to 25 ml of theseed medium described below in 250 ml corner-baffled flasks, followed byshaking culture at 200 rpm at 30° C. for 20 hours. 1 ml of the seedculture solution was added to a 250 ml corner-baffled flask including 24ml of the production medium described below, followed by shaking cultureat 200 rpm at 30° C. for 72 hours. The respective compositions of theseed medium and the production mediums are described below.

<Seed Medium (pH 7.0)>

glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, a KH₂PO₄ 4 g,K₂HPO₄ 8 g, MgSO₄.7(H₂O) 0.5 g, biotin 100 μg, thiamine HCI 1000 μg,calcium pantothenate 2000 μg, nicotinamide 2000 μg (with reference to 1L of distilled water).

<Production Medium (pH 7.0)>

glucose 100 g, (NH₄)₂SO₄ 40 g, soy protein 2.5 g, corn steep solids 5 g,urea 3 g, KH₂PO₄ 1 g, MgSO₄.7(H₂O) 0.5 g, biotin 100 μg, thiamine HCI1000 μg, calcium pantothenate 2000 μg, nicotinamide 3000 μg, CaCO₃ 30 g(with reference to 1 L of distilled water).

After the culturing, the L-lysine concentration was measured using HPLC.When acetate was not added, the L-lysine concentration in the culturesolutions of Corynebacterium glutamicum KCCM11016P,KCCM11016P::aceE1-PaceA, and KCCM11016P::aceE2-PaceA is shown in Table1.

TABLE 1 Variation of L-lysine production (acetate not added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 43.5 43.1 43.4KCCM11016P::aceE1-PaceA 43.7 43.2 43.6 KCCM11016P::aceE2-PaceA 43.3 43.443.7

In addition, the strains were cultured by the same method except that 5g/L of acetate was added to the production medium to compare theL-lysine production. The L-lysine concentration in the culture solutionsis shown in Table 2.

TABLE 2 Variation of L-lysine production (5 g/L of acetate added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 45.6 45.3 45.8KCCM11016P::aceE1-PaceA 47.2 47.1 47.4 KCCM11016P::aceE2-PaceA 46.7 46.547.0

As shown in Table 1, in the absence of acetate, the L-lysineproductivity of KCCM11016P::aceE1-PaceA and KCCM11016P::aceE2-PaceAstrains was not different from that of the parent strain KCCM11016P.

However, as shown in Table 2, in the presence of acetate, the L-lysineproductivity of the KCCM11016P::aceE1-PaceA strain was over 3.6% higherthan that of the parent strain KCCM11016P, and that of theKCCM11016P::aceE2-PaceA strain was over 2.5% higher than that of theparent strain KCCM11016P.

In addition, comparison of the KCCM11016P::aceE1-PaceA strain and theKCCM11016P::aceE2-PaceA strain shows that the L-lysine production of theKCCM11016P::aceE1-PaceA strain in which the aceA promoter was insertedto the upstream of the aceE gene transcription terminator, which wasbetween the stop codon and the upstream of the transcription terminator,was more effective. It indicates that the region between the stop codonand the upstream of the transcription terminator may be used to inhibitgene expression more effectively.

Example 5: Preparation of pDZ-aceE-Ppta Vector for Inhibiting aceE GeneExpression

Acetate kinase (ackA, NCgl2656) and phosphotransacetylase (pta,NCgl2657), which are enzymes that are involved in an acetate metabolicprocess, form an operon, and the expression thereof is strengthened inthe presence of acetate. Therefore, when the promoters of the genes areused, the expression of a gene may be specifically induced in thepresence of acetate.

In Example 5, to inhibit the expression of the aceE gene in the presenceof acetate, a vector for using a pta-ack operon promoter that was thepta gene upstream promoter region was prepared.

To inhibit the expression of the aceE gene, a vector which may comprisea pta gene promoter in the downstream of the aceE gene stop codon, whichis between the stop codon and the upstream of the transcriptionterminator, was constructed so that the transcription from pta genepromoter may occur in a direction opposite to the original direction ofthe aceE gene transcription.

To obtain a fragment of a Corynebacterium glutamicum-derived aceE gene,the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used asa template to prepare a pDZ-aceE1 vector by the same method as Example2.

To obtain a promoter fragment of a Corynebacterium glutamicum-derivedpta gene, primers, which were designed to have an SpeI restrictionenzyme recognition site at a 5′ end and at a 3′ end of the fragment (SEQID NO: 15 and SEQ ID NO: 16), were synthesized. The chromosomal DNA ofCorynebacterium glutamicum KCCM11016P as a template, and the synthesizedprimers were used to perform a PCR to amplify a promoter region of about340 base pairs being represented by a nucleotide sequence of SEQ ID NO:2. The PCR amplification product and a DNA fragment that was obtained bytreating a pDZ-aceE1 vector with a SpeI restriction enzyme were clonedby using an In-fusion Cloning Kit (TAKARA, JP) to prepare apDZ-aceE1-Ppta vector.

SEQ ID NO: 15: Ppta-P7F 5′-gtcccttgagactagtctttgctggggtcagatttg-3′SEQ ID NO: 16: Ppta-P7R 5′-ctgaggaataactagtacatcgcctttctaatttc-3′

Example 6: Preparation of Strains in Which pta Gene Promoter is Insertedto Downstream of aceE Gene and Comparison of Lysine Productivity Thereof

The Corynebacterium glutamicum KCCM11016P was transformed with thepDZ-aceE1-Ppta vector prepared in Example 5 by the same method asExample 3. The strain in which a pta gene promoter was inserted to thedownstream of the aceE gene stop codon on the chromosome so that thetranscription may occur in a direction opposite to the originaldirection of the aceE gene transcription was selected by performing aPCR to obtain an L-lysine-producing strain, which was named asKCCM11016P::aceE1-Ppta. The prepared KCCM11016P::aceE1-Ppta strain wasverified that the nucleotide sequence of target region obtained wasanalyzed by performing a PCR using SEQ ID NO: 3 and SEQ ID NO: 6 asprimers.

The prepared strain was cultured by the same method as Example 4, andthe concentration of L-lysine recovered from the culture solution wasmeasured. When acetate was not added, the L-lysine concentration in theculture solution is shown in Table 3.

TABLE 3 Variation of L-lysine production (acetate not added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 42.9 43.5 43.4KCCM11016P::aceE1-Ppta 43.2 43.3 43.6

In addition, the strain was cultured by the same method except that 5g/L of acetate was added to the production medium to compare theL-lysine production. The L-lysine concentration in the culture solutionis shown in Table 4.

TABLE 4 Variation of L-lysine production (5 g/L of acetate added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 45.5 45.7 45.3KCCM11016P::aceE1-Ppta 46.7 46.5 46.6

As shown in Table 3, in the absence of acetate, the L-lysineproductivity of the KCCM11016P::aceE1-Ppta strain was not different fromthat of the parent strain KCCM11016P.

However, as shown in Table 4, in the presence of acetate, the L-lysineproductivity of the KCCM11016P::aceE1-Ppta strain was over 2.4% higherthan that of the parent strain KCCM11016P.

In addition, since the L-lysine productivity of theKCCM11016P::aceE1-PaceA strain was higher than that of theKCCM11016P::aceE1-Ppta strain, in the presence of acetate, theexpression of a target gene may be inhibited more effectively by usingthe aceA gene promoter than by using the pta gene promoter.

Example 7: Preparation of Strains in Which aceA Gene Promoter isInserted to Downstream of aceE Gene

Three L-lysine-producing strains which were Corynebacterium glutamicumKFCC10750, KCCM10770P, and CJ3P were transformed respectively with thepDZ-aceE1-PaceA vector prepared in Example 2 by the same method asExample 3. The strains in which an aceA gene promoter was inserted tothe downstream of the aceE gene stop codon on the chromosome so that thetranscription may occur in a direction opposite to the originaldirection of the aceE gene transcription was selected by performing aPCR. The obtained three L-lysine-producing strains wereKFCC10750::aceE1-PaceA, KCCM10770P::aceE1-PaceA, and CJ3P::aceE1-PaceA.The prepared strains were verified that the nucleotide sequence oftarget region obtained was analyzed by performing a PCR using SEQ ID NO:3 and SEQ ID NO: 6 as primers.

The prepared strains were cultured by the same method as Example 4, andthe concentration of L-lysine recovered from the culture solutions wasmeasured. When acetate was not added, the L-lysine concentration in theculture solutions is shown in Table 5.

TABLE 5 Variation of L-lysine production (acetate not added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KFCC10750 38.3 38.0 38.4KFCC10750::aceE1- 38.6 38.2 38.3 PaceA KCCM10770P 47.5 47.3 47.6KCCM10770P::aceE1- 47.3 47.7 47.5 PaceA CJ3P 8 8.4 8.3 CJ3P::aceE1-PaceA8.2 8.1 8.5

In addition, the strains were cultured by the same method except that 5g/L of acetate was added to the production medium to compare theL-lysine production. The L-lysine concentration in the culture solutionsis shown in Table 6.

TABLE 6 Variation of L-lysine production (5 g/L of acetate added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KFCC10750 39.3 39.5 39.2KFCC10750::aceE1-PaceA 41.3 41.6 41.0 KCCM10770P 47.5 47.3 47.6KCCM10770P::aceE1- 49.0 48.6 48.8 PaceA CJ3P 8 8.4 8.3 CJ3P::aceE1-PaceA9.5 9.7 9.4

As shown in Table 5, in the absence of acetate, the L-lysineproductivity of the three KFCC10750::aceE1-PaceA,KCCM10770P::aceE1-PaceA, CJ3P::aceE1-PaceA strains was not differentfrom that of the parent strain.

However, as shown in Table 6, in the presence of acetate, the L-lysineproductivity of the KFCC10750::aceE1-PaceA strain was 5% higher thanthat of the parent strain, that of the KCCM10770P::aceE1-PaceA strainwas 2.8% higher than that of the parent strain, and that of theCJ3P::aceE1-PaceA strain was 15% higher than that of the parent strain.

Example 8: Preparation of Vector for Inhibiting hom Gene Expression

L-threonine biosynthetic pathway using a same substrate as L-lysinebiosynthetic pathway may be weakened to increase L-lysine productivity.An example of the methods of weakening L-threonine biosynthetic pathwayis to decrease the enzymatic activity of homoserine dehydrogenase (hom,NCgl1136) which produces homoserine from aspartate.

In Corynebacterium glutamicum, the hom gene forms hom-thrB operon withthrB gene, and the transcription terminator of the hom gene exists inthe downstream of the thrB gene. It was reported that a promoter existsin the upstream of the hom-thrB operon that is the upstream of the homgene. In addition, it was reported a second promoter exists in theupstream of the thrB gene operon (Mateos et al., J Bacteriol,176:7362-7371, 1994). Therefore, an aceA gene promoter was inserted tothe downstream of the hom gene stop codon so that the trascription fromthis promoter may occur in a direction opposite to the originaldirection of the hom gene transcription in order to selectively inhibitthe expression of the hom gene in the presence of acetate. To maintainthe expression of the thrB gene, a second promoter sequence was added tothe upstream of thrB gene ORF.

In Example 8, a recombinant vector was prepared by inserting an aceAgene promoter between the downstream of the hom gene stop codon and theupstream of the thrB gene.

To obtain a fragment of a Corynebacterium glutamicum-derived hom gene,the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used asa template, and primers, which were designed to have an XbaI restrictionenzyme recognition site at a 5′ end of the fragment and an SpeIrestriction enzyme recognition site at a 3′ end of the fragment (SEQ IDNOs: 17 and 18), were synthesized. A PCR was performed using thesynthesized primers to obtain a DNA fragment including 300 base pairsbetween the 1039th nucleotide from the hom gene start codon and the1338th nucleotide which was a stop codon. Even when an aceA is insertedbetween the hom-thrB operons, the expression of the thrB gene should bemaintained. Therefore, when the DNA fragment including the 300 basepairs of the hom gene stop codon downstream was prepared, primers whichwere designed to further add a thrB promoter sequence of 32 base pairsto a 5′ side (SEQ ID NOs: 19 and 20) were synthesized. A PCR wasperformed using these primers (SEQ ID NOs: 19 and 20) to obtain a DNAfragment of 334 base pairs having an SpeI restriction enzyme recognitionsite at a 5′ end of the fragment and an XbaI restriction enzymerecognition site at a 3′ end of the fragment. The PCR was performedunder the same conditions as Example 2.

The two PCR amplification products and a pDZ vector for chromosomalintroduction that had already been prepared by cleaving with an XbaIrestriction enzyme were cloned by using an In-fusion Cloning Kit(TAKARA, JP) to prepare a pDZ-hom vector.

SEQ ID NO: 17: hom-h1F 5′-ccggggatcctctagaccaggtgagtccacctacg-3′SEQ ID NO: 18: hom-h1R 5′-gaggcggatcactagtttagtccctttcgaggcgg-3′SEQ ID NO: 19: hom-h2F 5′-actagtgatccgcctcgaaagggac-3′SEQ ID NO: 20: hom-h2R 5′-gcaggtcgactctagagactgcggaatgttgttgtg-3′

To obtain a promoter fragment of a Corynebacterium glutamicum-derivedaceA gene, primers which were designed to have an SpeI restrictionenzyme recognition site at a 5′ end and at a 3′ end of the fragment (SEQID NOs: 21 and 22) were synthesized. The PCR was performed using thechromosomal DNA of Corynebacterium glutamicum KCCM11016P as a template,and the synthesized primers to amplify a promoter region of about 500base pairs being represented by a nucleotide sequence of SEQ ID NO: 1.The PCR amplification product and a DNA fragment that was obtained bytreating a pDZ-hom vector with a SpeI restriction enzyme were cloned byusing an In-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-hom-PaceAvector.

SEQ ID NO: 21: PaceA-h3F 5′-gaggcggatcactagtagcactctgactacctctg-3′SEQ ID NO: 22: PaceA-h3R 5′-aagggactaaactagtttcctgtgcggtacgtggc-3′

Example 9: Preparation of Strains to Which aceA Gene Promoter isInserted to Downstream of hom Gene and Comparison of Lysine ProductivityThereof

The Corynebacterium glutamicum KCCM11016P that is an L-lysine-producingstrain was transformed with the pDZ-hom-PaceA vector prepared in Example8 by the same method as Example 3. The strain in which an aceA genepromoter was inserted to the downstream of the hom gene stop codon onthe chromosome so that the transcription may occur in a directionopposite to the original direction of the hom gene transcription wasselected by performing a PCR to obtain an L-lysine-producing strain,which was named as KCCM11016P::hom-PaceA. The preparedKCCM11016P::hom-PaceA strain was verified that the nucleotide sequenceof target region obtained was analyzed by performing a PCR using SEQ IDNO: 17 and SEQ ID NO: 20 as primers.

The Corynebacterium glutamicum KCCM11016P strain which was used as aparent strain and the prepared KCCM11016P::hom-PaceA strain werecultured by the same method as Example 4, and the concentration ofL-lysine recovered from the culture solutions was measured. When acetatewas not added, the L-lysine concentration in the culture solutions ofthe Corynebacterium glutamicum KCCM11016P strain and theKCCM11016P::hom-PaceA is shown in Table 7.

TABLE 7 Variation of L-lysine production (acetate not added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 43.2 43.3 43.6KCCM11016P::hom-PaceA 43.3 43.6 43.4

In addition, the strains were cultured by the same method except that 5g/L of acetate was added to the production medium to compare theL-lysine production. The L-lysine concentration in the culture solutionsis shown in Table 8.

TABLE 8 Variation of L-lysine production (5 g/L of acetate added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 44.9 45.6 45.2KCCM11016P::hom-PaceA 46.3 46.6 46.4

As shown in Table 7, in the absence of acetate, the L-lysineproductivity of the KCCM11016P::hom-PaceA strain was not different fromthat of the parent strain KCCM11016P.

However, as shown in Table 8, in the presence of acetate, the L-lysineproductivity of the KCCM11016P::hom-PaceA strain was over 2.6% higherthan that of the parent strain KCCM11016P.

Example 10: Preparation of Vector for Inhibiting murE Gene Expression

UDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase (murE,NCgl2083) uses meso-2,6-diaminopimelate, which is a precursor for lysinebiosynthesis, in somatic synthesis. Weakening of the murE gene activitymay decrease the inflow of carbon sources to somatic synthesis andincrease the inflow of carbon sources to a lysine biosynthetic pathwayto increase lysine production.

In Corynebacterium glutamicum, the murE gene (NCgl2083) forms an operonwith seven genes from NCgl2076 to NCgl2082. The transcription of theoperon starts from the NCgl2083 murE in the direction of the NCgl2076gene. So, a transcription terminator exists in the downstream of theNCgl2076 gene. Therefore, an aceA gene promoter was inserted to thedownstream of the murE gene stop codon so that the transcription mayoccur in a direction opposite to the original direction of the murE genetranscription in order to selectively inhibit the expression of the murEgene in the presence of acetate. To maintain the expression of the otherseven genes except the murE gene which is located in the first region ofthe operon, a murE operon promoter was further added to the upstream ofthe NCgl2082 gene ORF.

In Example 10, a recombinant vector was prepared by inserting an aceAgene promoter to the downstream of the murE gene stop codon.

To obtain a fragment of a Corynebacterium glutamicum-derived murE gene,the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used asa template, and primers, which were designed to have an XbaI restrictionenzyme recognition site at a 5′ end of the fragment and an Xholrestriction enzyme recognition site at a 3′ end of the fragment (SEQ IDNOs: 23 and 24), were synthesized. A PCR was performed using thesynthesized primers to obtain a DNA fragment including 300 base pairsbetween the 1267th nucleotide from the murE gene start codon and the1566th nucleotide which was a stop codon. In addition, primers, whichwere designed to have an Xhol restriction enzyme recognition site at a5′ end of the fragment and an XbaI restriction enzyme recognition siteat a 3′ end of the fragment (SEQ ID NOs: 25 and 26), were synthesized. APCR was performed using the synthesized primers to obtain a DNA fragmentincluding 292 base pairs from the 10th nucleotide in the downstream ofthe murE gene stop codon. The PCR was performed under the sameconditions as Example 2. The two PCR amplification products and a pDZvector for chromosomal introduction that had already been prepared bycleaving with an XbaI restriction enzyme were cloned by using anIn-fusion Cloning Kit (TAKARA, JP) to prepare a pDZ-murE vector.

SEQ ID NO: 23: mur-m1F 5′-ccggggatcctctagaaaccctcgttcagaggtgc-3′SEQ ID NO: 24: mur-m1R 5′-ttgtgatcatctcgagctatccttcttccgtagtaag-3′SEQ ID NO: 25: mur-m2F 5′-ag ctcgagatgatcacaatgacccttgg-3′SEQ ID NO: 26: mur-m2R 5′-gcaggtcgactctagacatgagcataaatgtcagc-3′

To obtain a fragment of a Corynebacterium glutamicum-derived aceA gene,primers, which were designed to have an Xhol restriction enzymerecognition site at a 5′ end of the fragment (SEQ ID NOs: 27 and 28),were synthesized. The chromosomal DNA of Corynebacterium glutamicumKCCM11016P as a template, and the synthesized primers were used toperform a PCR to amplify a promoter region of about 500 base pairs beingrepresented by a base sequence of SEQ ID NO: 1. In addition, to obtain apromoter region of a Corynebacterium glutamicum-derived murE operon, thechromosomal DNA of Corynebacterium glutamicum KCCM11016P was used as atemplate, and primers, which were designed to have an Xhol restrictionenzyme recognition site at a 3′ end of the fragment (SEQ ID NOs: 29 and30), were synthesized. A PCR was performed using the synthesized primersto obtain a DNA fragment including 300 base pairs in the upstream ofmurE gene ORF.

The two PCR amplification products and the DNA fragment obtained bytreating the pDZ-murE vector with Xhol restriction enzyme were cloned byusing an In-fusion Cloning Kit (TAKARA, JP) to prepare apDZ-murE-PaceA-PmurE vector.

SEQ ID NO: 27: mur-m3F 5′-tcatcagcagcactctgactacctctg-3′SEQ ID NO: 28: mur-m3R 5′-agaaggatagctcgagttcctgtgcggtacgtggc-3′SEQ ID NO: 29: mur-m4F 5′-agagtgctgctgatgatcctcgatttg-3′SEQ ID NO: 30: mur-m4R 5′-ttgtgatcatctcgagggttttctctcctccacagg-3′

Example 11: Preparation of Strains to Which aceA Gene Promoter isInserted to Downstream of murE Gene and Comparison of LysineProductivity Thereof

The Corynebacterium glutamicum KCCM11016P was transformed with thepDZ-murE-PaceA-PmurEvector prepared in Example 10 by the same method asExample 3.

The strain in which an aceA gene promoter was inserted to the downstreamof the murE gene stop codon on the chromosome so that the transcriptionmay occur in a direction opposite to the original direction of the murEgene transcription was selected by performing a PCR to obtain anL-lysine-producing strain, which was named asKCCM11016P::murE-PaceA-PmurE. The prepared KCCM11016P::murE-PaceA-PmurEstrain was verified that the nucleotide sequences of target regionobtained was analyzed by performing a PCR using SEQ ID NO: 23 and SEQ IDNO: 26 as primers.

The Corynebacterium glutamicum KCCM11016P strain which was used as aparent strain and the prepared KCCM11016P::murE-PaceA-PmurE strain werecultured by the same method as Example 4, and the concentration ofL-lysine recovered from the culture solutions was measured. When acetatewas not added, the L-lysine concentration in the culture solutions ofthe Corynebacterium glutamicum KCCM11016P strain and theKCCM11016P::murE-PaceA-PmurE strain is shown in Table 9.

TABLE 9 Variation of L-lysine production (acetate not added) Lysine(g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 43.5 43.9 44.0KCCM11016P::murE-PaceA-PmurE 43.7 44.1 43.8

In addition, the strains were cultured by the same method except that 5g/L of acetate was added to the production medium to compare theL-lysine production. The L-lysine concentration in the culture solutionsis shown in Table 10.

TABLE 10 Variation of L-lysine production (5 g/L of acetate added)Lysine (g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11016P 45.2 45.6 45.3KCCM11016P::murE-PaceA-PmurE 46.6 46.9 46.5

As shown in Table 9, in the absence of acetate, the L-lysineproductivity of the KCCM11016P::murE-PaceA-PmurE strain was notdifferent from that of the parent strain KCCM11016P.

However, as shown in Table 10, in the presence of acetate, the L-lysineproductivity of the KCCM11016P::murE-PaceA-PmurE strain was over 2.8%higher than that of the parent strain KCCM11016P.

Example 12: Preparation of Vector for Inhibiting dapA Gene Expression

L-lysine biosynthetic pathway using the same substrate as L-threoninebiosynthetic pathway may be weakened to increase L-threonineproductivity. An example of the methods of weakening L-lysinebiosynthetic pathway is to decrease the enzymatic activity ofdihydrodipicolinate synthase (dapA, NCgl1896) which is involved in theproduction of lysine from aspartate.

In Corynebacterium glutamicum, the dapA gene forms dapA-ORF4 operon withORF4 (NCgl1895) gene, and thus the transcription terminator of the dapAgene exists in the downstream of the ORF4 gene. In addition, it wasreported that a promoter exists in the upstream of the dapA-ORF4 operonthat is the upstream of the dapA gene, and a second promoter exists inthe upstream of the ORF4 gene (Patek et al., Biotechnology letters,19:1113-1117, 1997). Therefore, an aceA gene promoter was inserted tothe downstream of the dapA gene stop codon so that the transcription mayoccur in a direction opposite to the original direction of the dapA genetranscription in order to selectively inhibit the expression of the dapAgene in the presence of acetate. To maintain the expression of the ORF4gene, a promoter region sequence of about 100 base pairs in the ORF4gene upstream was added to the ORF upstream of the ORF4 gene.

In Example 12, a recombinant vector was prepared by inserting an aceAgene promoter to the downstream of the dapA gene stop codon.

To obtain a fragment of a Corynebacterium glutamicum-derived dapA gene,the chromosomal DNA of Corynebacterium glutamicum KCCM11016P was used asa template, and primers, which were designed to have an XbaI restrictionenzyme recognition site at a 5′ end of the fragment and an SpeIrestriction enzyme recognition site at a 3′ end of the fragment (SEQ IDNOs: 31 and 32), were synthesized. A PCR was performed using thesynthesized primers to obtain a DNA fragment including 301 base pairsbetween the 606th nucleotide from the dapA gene start codon and the906th nucleotide which was a stop codon. In addition, primers, whichwere designed to have an SpeI restriction enzyme recognition site at a5′ end of the fragment and an XbaI restriction enzyme recognition siteat a 3′ end of the fragment (SEQ ID NOs: 33 and 34), were synthesized.Through a PCR, a DNA fragment which further includes a promoter regionsequence of about 100 base pairs between the 809th nucleotide from thedapA gene start codon and the 2nd nucleotide in the stop codondownstream, to maintain the expression of the ORF4 gene, and 213 basepairs in the dapA gene stop codon downstream was obtained. The PCR wasperformed under the same conditions as Example 2. The two PCRamplification products and a pDZ vector for chromosomal introductionthat had already been prepared by cleaving with an XbaI restrictionenzyme were cloned by using an In-fusion Cloning Kit (TAKARA, JP) toprepare a pDZ-dapA vector.

SEQ ID NO: 31: dapA-d1F 5′-ccggggatcctctaga tgtttggcttgctttgggc-3′SEQ ID NO: 32: dapA-d1R 5′-gttgatgcactagtttatagaactccagcttt-3′SEQ ID NO: 33: dapA-d2F 5′-ttctataaactagtgcatcaacgtaggagatcc-3′SEQ ID NO: 34: dapA-d2R 5′-gcaggtcgactctagacgttctgggaaccctgag-3′

To obtain a promoter fragment of a Corynebacterium glutamicum-derivedaceA gene, primers, which were designed to have an SpeI restrictionenzyme recognition site at a 5′ end and at a 3′ end of the fragment (SEQID NOs: 35 and 36), were synthesized. The PCR was performed using thechromosomal DNA of Corynebacterium glutamicum KCCM11016P as a templateand the synthesized primers to amplify a promoter region of about 500base pairs being represented by a nucleotide sequence of SEQ ID NO: 1.The PCR amplificated product and a DNA fragment,which was obtained bytreating a pDZ-dapA vector with a SpeI restriction enzyme, were clonedby using an In-fusion Cloning Kit (TAKARA, JP) to prepare apDZ-dapA-PaceA vector.

SEQ ID NO: 35: PaceA-d3F 5′-acgttgatgc actagt agcactctgactacctctg-3′SEQ ID NO: 36: PaceA-d3R 5′-agttctataa actagt ttcctgtgcggtacgtggc-3′

Example 13: Preparation of Strains in Which aceA Gene Promoter isInserted to Downstream of dapA Gene and Comparison of ThreonineProductivity Thereof

To verify the effect of inhibiting the dapA gene expression in anL-threonine-producing strain, the pDZ-dapA-PaceA vector prepared inExample 12 was transformed by the same method as Example 3 into aCorynebacterium glutamicum KCCM11222P (Korean Patent NO: 2013-0061570)strain that is an L-threonine-producing strain. The strain in which anaceA gene promoter was inserted to the downstream of the dapA gene stopcodon on the chromosome so that the transcription may occur in adirection opposite to the original direction of the dapA genetranscription was selected by performing a PCR, which was named asKCCM11222P::dapA-PaceA. The prepared KCCM11222P::dapA-PaceA strain wasverified that the nucleotide sequence of target region obtained wasanalyzed by performing a PCR by using SEQ ID NO: 31 and SEQ ID NO: 34 asprimers.

The Corynebacterium glutamicum KCCM11222P strain, which was used as aparent strain, and the prepared KCCM11222P::dapA-PaceA strain werecultured by the method described below.

Each of the strains was respectively inoculated 25 ml of a seed mediumin 250 ml corner-baffled flasks, followed by shaking culture at 200 rpmat 30° C. for 20 hours. Afterward, 1 ml of the seed culture solution wasadded to a 250 ml corner-baffled flask including 24 ml of a productionmedium, followed by shaking culture at 200 rpm at 30° C. for 48 hours at200 rpm. The respective compositions of the seed medium and theproduction mediums are described below.

<Seed Medium (pH 7.0)>

glucose 20 g, peptone 10 g, yeast extract 5 g, urea 1.5 g, KH₂PO₄ 4 g,K₂HPO₄ 8 g, MgSO₄.7(H₂O) 0.5 g, biotin 100 μg, thiamine HCI 1000 μg,calcium pantothenate 2000 μg, nicotinamide 2000 μg (with reference to 1L of distilled water).

<Production Medium (pH 7.0)>

glucose 100 g, (NH₄)₂SO₄ 20 g, soy protein 2.5 g, corn steep solids 5 g,urea 3 g, KH₂PO₄ 1 g, MgSO₄.7(H₂O) 0.5 g, biotin 100 μg, thiamine HCI1000 μg, calcium pantothenate 2000 μg, nicotinamide 3000 μg, CaCO₃ 30 g(with reference to 1 L of distilled water).

After the culturing, L-threonine concentration in the culture solutionwas measured by HPLC. When acetate was not added, the L-threonineconcentration in the culture solutions of Corynebacterium glutamicumKCCM11222P and KCCM11222P::dapA-PaceA is shown in Table 11.

TABLE 11 Variation of L-threonine production (acetate not added)Threonine (g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11222P 7.0 6.9 7.2KCCM11222P::dapA-PaceA 7.1 7.3 7.0

In addition, the strains were cultured by the same method except that 5g/L of acetate was added to the production medium to compare theL-threonine production. The L-threonine concentration in the culturesolutions is shown in Table 12.

TABLE 12 Variation of L-threonine production (5 g/L of acetate added)Threonine (g/L) Strain Batch 1 Batch 2 Batch 3 KCCM11222P 7.6 7.4 7.7KCCM11222P::dapA-PaceA 11.2 11.5 11.4

As shown in Table 11, in the absence of acetate, the L-threonineproductivity of the KCCM11222P::dapA-PaceA strain was not different fromthat of the parent strain KCCM11222P.

However, as shown in Table 12, in the presence of acetate, theL-threonine productivity of the KCCM11222P::dapA-PaceA strain was over50% higher than that of the parent strain KCCM11222P.

ACCESSION NUMBER

Research Center Name: Korean Collection for Type Cultures(International)

Accession Number: KCCM11432P

Accession Date: Jun. 12, 2013

As described above, according to the one or more of the aboveembodiments of the present invention, an acetate-inducible promoter maybe used to effectively produce L-amino acids, since a target L-aminoacid may be produced in a high yield by weakening the expression of atarget gene by adding acetate at an appropriate time.

It should be understood that the exemplary embodiments described thereinshould be considered in a descriptive sense only and not for purposes oflimitation. Descriptions of features or aspects within each embodimentshould typically be considered as available for other similar featuresor aspects in other embodiments.

While one or more embodiments of the present invention have beendescribed with reference to the figures, it will be understood by thoseof ordinary skill in the art that various changes in form and detailsmay be made therein without departing from the spirit and scope of thepresent invention as defined by the following claims.

What is claimed is:
 1. A method of producing a target L-amino acids, themethod comprising culturing a recombinant coryneform microorganism in amedium, wherein the recombinant coryneform microorganism is capable ofproducing a target L-amino acid and is transformed by inserting anacetate-inducible promoter downstream of a stop codon of a target genein a chromosome such that transcription from the acetate-induciblepromoter occurs in a direction opposite to the direction of the targetgene transcription, wherein the target gene is a gene located at abranch point of a biosynthetic pathway of the target L-amino acid andencoding an enzyme involved in byproduct production, wherein the targetL-amino acid is L-threonine, L-methionine, L-isoleucine, L-lysine,L-alanine, or L-valine.
 2. The method according to claim 1, wherein thedownstream of the stop codon is between the stop codon and an upstreamof a transcription terminator of the target gene.
 3. The methodaccording to claim 1, wherein the acetate-inducible promoter is anucleotide sequence of SEQ ID NO: 1 or SEQ ID NO:
 2. 4. The methodaccording to claim 1, wherein the target gene is at least one geneselected from the group consisting of a gene encoding a pyruvatedehydrogenase subunit E1, a gene encoding homoserine dehydrogenase, anda gene encoding aUDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase; andthe target L-amino acid is L-lysine.
 5. The method according to claim 1,wherein the target gene is at least one gene selected from the groupconsisting of a gene encoding a pyruvate dehydrogenase subunit E1 and agene encoding dihydrodipicolinate synthase; and the target L-amino acidis L-threonine.
 6. The method according to claim 1, wherein the targetgene is at least one gene selected from the group consisting of a geneencoding a pyruvate dehydrogenase subunit E1, a gene encoding homoserinedehydrogenase, a gene encoding aUDP-N-acetylmuramoylalanyl-D-glutamate-2,6-diaminopimelate ligase, agene encoding a homoserine kinase, and a gene encodingdihydrodipicolinate synthase.
 7. The method according to claim 1,further comprising adding acetate during the culturing to cause atranscription from the acetate-inducible promoter in a directionopposite to the direction of the target gene transcription so that RNApolymerase complexes conflict with each other to weaken the expressionof the target gene, and to strengthen the target L-amino acid productioncapability of the recombinant coryneform microorganism.